Blue:RNA-Seq Experiments:11132013: Difference between revisions
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::#Remove supernatant and wash 2x with 1ml 80% EtOH | ::#Remove supernatant and wash 2x with 1ml 80% EtOH | ||
::#Air Dry 10min or until cracking first occurs | ::#Air Dry 10min or until cracking first occurs | ||
::#Resuspend beads in | ::#Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube | ||
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|98C 15s -> 58C 20s -> 72C 30s||||||||||6x | |98C 15s -> 58C 20s -> 72C 30s||||||||||6x | ||
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|98C 10s -> 72C 20s|||||||||| | |98C 10s -> 72C 20s||||||||||9x | ||
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|72C, 5min | |72C, 5min |
Revision as of 21:41, 14 November 2013
RNA-Seq Library Information
Library ID | Samples | Input RNA | TSO Primer | Bar Code Primers | Bar Code Primer 2 | Type of Seq | Reads | Library Conc |
BL_ | UHRR/ERCC | 10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP | TSO.r06 | T20VN_PE_R | N2.id90 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP/3'dATP (1:0.1) | TSO.r06 | T20VN_PE_R | N2.id91 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP/dATP (1:1) | TSO.r06 | T20VN_PE_R | N2.id92 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP | TSO.r06 | T20VN_PE_R | N2.id93 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/3'dATP (1:0.1) | TSO.r06 | T20VN_PE_R | N2.id94 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/dATP (1:1) | TSO.r06 | T20VN_PE_R | N2.id95 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP then add 3'dATP | TSO.r06 | T20VN_PE_R | N2.id96 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10ng - RNase3 (Affy buffer)/Affymetrix PAP 10min ATP then add 3'dATP | TSO.r06 | T20VN_PE_R | N2.id97 | totoRNAseq |
Purpose
- Test original totoRNAseq protocol (using RNaseIII buffer and 5xRT for RnaseIII/PAP treatment) without heating to 65C to inactivate enzymes. RNaseIII inactivation by RNase inhibitor addition and PAP limitation by 3'dATP or dATP addition
- Testing difference of Ambion PAP and Affymetrix PAP (note that Affymetrix PAP showed significant A tailing of TSO and was completely inhibited by 3'dATP)
RNAseIII-Fragmented Library Preparation
- RNA
Volume UHRR 20ng/ul 0.5ul ERCC 1:10E3 0.2ul 1uM T20VN 0.1ul dH2O 0.2ul Total 1ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1 min
Volume 10x RNaseIII buffer or 5x PAP buffer 0.2ul or 0.4ul RNase III 0.1ul dH20 0.7ul or 0.5ul Total 2ul
- Incubate @ 37C 5 min
- 3' Tailing
Volume fRNA 2ul 5x RT buffer or 5x Affy PAP Buffer 0.5ul or 0.4ul 1mM ATP (or mix) 0.5ul 2U/ul PAP 0.2ul 40U/ul Rnase Inhibitor 0.2ul dH2O 0.6ul or 0.7ul Total 4ul
- Incubate @ 37C 10 min
- Add 1ul of 1uM T20VN or 0.5ul 2uM T20VN/0.05uM 3'dATP
- Reverse Transcription
Volume PAP RNA 5ul (or 4.5ul) 5xRT Buffer 1.5ul (or 2ul) 10mM dNTP Mix 1ul 100mM DTT 0.25ul Betaine (5M) 2ul Smarter MMLV (200U/ul) 0.25ul Total 10ul
- Incubate @ 42C 30 min
- Beads Purification
- Add 15ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube
- TSO
Volume Purified cDNA 3ul 5x RT buffer [MMLV] 2ul 10mM dNTP mix 1ul 100mM DTT 0.25ul 10uM TSO 1ul Betaine (5M) 2ul RNase Inhibitor 0.25ul Smarter MMLV 0.5ul Total 10ul
- Incubate @42C 10min
- Incubate @70C 10min
- PCR (PhusionHF)
Volume TSO Reaction 10ul 2x Buffer 25ul 10uM P1-STRT 1ul 10uM PCR_R_N2_id 1ul H2O 13ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 9x 72C, 5min
Results of Amplification
- Run 5ul on gel:
Final Library Preparation
- Beads purified twice at (0.8x), re-suspend in 10ul